General anaesthetics do not impair developmental expression of the KCC2 potassium-chloride cotransporter in neonatal rats during the brain growth spurt.

BACKGROUND: The developmental transition from depolarizing to hyperpolarizing γ-aminobutyric acid-mediated neurotransmission is primarily mediated by an increase in the amount of the potassium-chloride cotransporter KCC2 during early postnatal life. However, it is not known whether early neuronal activity plays a modulatory role in the expression of total KCC2 mRNA and protein in the immature brain. As general anaesthetics are powerful modulators of neuronal activity, the purpose of this study was to explore how these drugs affect KCC2 expression during the brain growth spurt. METHODS: Wistar rat pups were exposed to either a single dose or 6 h of midazolam, propofol, or ketamine anaesthesia at postnatal days 0, 5, 10, or 15. KCC2 expression was assessed using immunoblotting, immunohistochemistry, or quantitative polymerase chain reaction analysis up to 3 days post-exposure in the medial prefrontal cortex. RESULTS: There was a progressive and steep increase in the expression of KCC2 between birth and 2 weeks of age. Exposure to midazolam, propofol, or ketamine up to 6 h at any investigated stages of the brain growth spurt did not influence the expression of this cotransporter protein. CONCLUSION: I.V. general anaesthetics do not seem to influence developmental expression of KCC2 during the brain growth spurt.

Anaesthetic neurotoxicity and neuroplasticity: an expert group report and statement based on the BJA Salzburg Seminar.

Although previously considered entirely reversible, general anaesthesia is now being viewed as a potentially significant risk to cognitive performance at both extremes of age. A large body of preclinical as well as some retrospective clinical evidence suggest that exposure to general anaesthesia could be detrimental to cognitive development in young subjects, and might also contribute to accelerated cognitive decline in the elderly. A group of experts in anaesthetic neuropharmacology and neurotoxicity convened in Salzburg, Austria for the BJA Salzburg Seminar on Anaesthetic Neurotoxicity and Neuroplasticity. This focused workshop was sponsored by the British Journal of Anaesthesia to review and critically assess currently available evidence from animal and human studies, and to consider the direction of future research. It was concluded that mounting evidence from preclinical studies reveals general anaesthetics to be powerful modulators of neuronal development and function, which could contribute to detrimental behavioural outcomes. However, definitive clinical data remain elusive. Since general anaesthesia often cannot be avoided regardless of patient age, it is important to understand the complex mechanisms and effects involved in anaesthesia-induced neurotoxicity, and to develop strategies for avoiding or limiting potential brain injury through evidence-based approaches.

New pool of cortical interneuron precursors in the early postnatal dorsal white matter.

The migration of cortical γ-aminobutyric acidergic interneurons has been extensively studied in rodent embryos, whereas few studies have documented their postnatal migration. Combining in vivo analysis together with time-lapse imaging on cortical slices, we explored the origin and migration of cortical interneurons during the first weeks of postnatal life. Strikingly, we observed that a large pool of GAD65-GFP-positive cells accumulate in the dorsal white matter region during the first postnatal week. Part of these cells divides and expresses the transcription factor paired box 6 indicating the presence of local transient amplifying precursors. The vast majority of these cells are immature interneurons expressing the neuronal marker doublecortin and partly the calcium-binding protein calretinin. Time-lapse imaging reveals that GAD65-GFP-positive neurons migrate from the white matter pool into the overlying anterior cingulate cortex (aCC). Some interneurons in the postnatal aCC express the same immature neuronal markers suggesting ongoing migration of calretinin-positive interneurons. Finally, bromodeoxyuridine incorporation experiments confirm that a small fraction of interneurons located in the aCC are generated during the early postnatal period. These results altogether reveal that at postnatal ages, the dorsal white matter contains a pool of interneuron precursors that divide and migrate into the aCC.

Excess of serotonin affects embryonic interneuron migration through activation of the serotonin receptor 6.

The discovery that a common polymorphism (5-HTTLPR, short variant) in the human serotonin transporter gene (SLC6A4) can influence personality traits and increase the risk for depression in adulthood has led to the hypothesis that a relative increase in the extracellular levels of serotonin (5-HT) during development could be critical for the establishment of brain circuits. Consistent with this idea, a large body of data demonstrate that 5-HT is a strong neurodevelopmental signal that can modulate a wide variety of cellular processes. In humans, serotonergic fibers appear in the developing cortex as early as the 10th gestational week, a period of intense neuronal migration. In this study we hypothesized that an excess of 5-HT could affect embryonic cortical interneuron migration. Using time-lapse videometry to monitor the migration of interneurons in embryonic mouse cortical slices, we discovered that the application of 5-HT decreased interneuron migration in a reversible and dose-dependent manner. We next found that 5-HT6 receptors were expressed in cortical interneurons and that 5-HT6 receptor activation decreased interneuron migration, whereas 5-HT6 receptor blockade prevented the migratory effects induced by 5-HT. Finally, we observed that interneurons were abnormally distributed in the cerebral cortex of serotonin transporter gene (Slc6a4) knockout mice that have high levels of extracellular 5-HT. These results shed new light on the neurodevelopmental alterations caused by an excess of 5-HT during the embryonic period and contribute to a better understanding of the cellular processes that could be modulated by genetically controlled differences in human 5-HT homeostasis.

Recruiting new neurons from the subventricular zone to the rat postnatal cortex: an organotypic slice culture model.

The neurogenic subventricular zone (SVZ) of the lateral ventricle is a potential source for neuronal replacement in the postnatal or adult neocortex after injury. Here we present a novel model system to directly explore the cellular mechanisms of this process. In order to visualize directed migration from the SVZ towards the cortex, we transplanted green fluorescent protein-labeled progenitor/stem cells into the SVZ of newborn rats. At 2 days after transplantation, we generated organotypic slice cultures and applied fluorescent time-lapse imaging to explore directly the migration and integration of donor cells into the host tissue for up to 2 weeks. Our studies revealed that subventricular grafts provide a significant number of immature neurons to neocortical regions. In the cortex, immature neurons first migrate radially towards the pial surface and then differentiate into GABAergic interneurons. We conclude that our model system presents a novel and effective experimental paradigm to evaluate the recruitment of SVZ-derived neurons into the postnatal cortex, a phenomenon that may represent a potential route for cortical repair.

Magnocellular vasopressinergic neurons in explant cultures are rescued from cell death by ciliary neurotrophic factor and leukemia inhibiting factor.

Selective death of magnocellular vasopressinergic neurons in the hypothalamus has been reported in cases of hereditary and idiopathic diabetes insipidus and after experimental lesions of the hypothalamo-neurohypophyseal pathway. To identify trophic factors that promote survival of these neurons, an in vitro model system was established in which organotypic cultures of the rat hypothalamic paraventricular nucleus were maintained in chemically-defined medium. We observe that the majority of magnocellular vasopressinergic neurons die in these cultures, while other cell populations such as corticotrophin-releasing factor producing parvicellular and oxytocin producing magnocellular cells retain a well preserved cytoarchitectonic organization. Degenerating vasopressinergic cells exhibit morphological signs of apoptosis and stained positively when analysed by the terminal deoxynucleotidyl transferase biotinylated dUTP nick end-labelling assay. Partial survival of vasopressinergic neurons occurred after co-culturing the paraventricular nucleus with neurohypophyseal explants, indicating that target-derived factors may be required for the survival of these neurons. Cell survival is dramatically increased by the administration of ciliary neurotrophic factor and leukemia inhibiting factor, but not by interleukin 6 or the members of the neurotrophin family. Reverse transcription-polymerase chain reaction followed by Southern analysis shows the presence of ciliary neurotrophic factor messenger RNA in the neurohypophysis. Thus, endogenous ciliary neurotrophic factor and leukemia inhibiting factor, produced by neurohypophyseal cells may function as a physiological survival factor for neurosecretory vasopressinergic neurons.

Excess of serotonin affects neocortical pyramidal neuron migration.

The serotonin transporter (SERT) is a key molecule involved in the homeostasis of extracellular levels of serotonin and is regulated developmentally. Genetic deletion of SERT in rodents increases extracellular levels of serotonin and affects cellular processes involved in neocortical circuit assembly such as barrel cortex wiring and cortical interneuron migration. Importantly, pharmacological blockade of SERT during brain development leads to phenotypes relevant to psychiatry in rodents and to an increased risk for autism spectrum disorders in humans. Furthermore, developmental adversity interacts with genetically-driven variations of serotonin function in humans and nonhuman primates to increase the risk for a variety of stress-related phenotypes. In this study, we investigate whether an excess of serotonin affects the migration of neocortical pyramidal neurons during development. Using in utero electroporation combined with time-lapse imaging to specifically monitor pyramidal neurons during late mouse embryogenesis, we show that an excess of serotonin reversibly affects the radial migration of pyramidal neurons. We further identify that the serotonin receptor 5-HT(6) is expressed in pyramidal neuron progenitors and that 5-HT(6) receptor activation replicates the effects of serotonin stimulation. Finally, we show that the positioning of superficial layer pyramidal neurons is altered in vivo in SERT knockout mice. Taken together, these results indicate that a developmental excess of serotonin decreases the migration speed of cortical pyramidal neurons, affecting a fundamental step in the assembly of neural circuits. These findings support the hypothesis that developmental dysregulation of serotonin homeostasis has detrimental effects on neocortical circuit formation and contributes to increased vulnerability to psychiatric disorders.

Potentially toxic effects of anaesthetics on the developing central nervous system.

A growing body of experimental evidence suggests that anaesthetics, by influencing GABAergic and glutaminergic neural signalling, can have adverse effects on the developing central nervous system. The biological foundation for this is that gamma-aminobutyric acid and glutamate could act non-synaptically, in addition to their role in neurotransmission in the adult brain, in the regulation of neuronal development in the central nervous system. These neurotransmitters and their receptors are expressed from very early stages of central nervous system development and appear to influence neural progenitor proliferation, cell migration and neuronal differentiation. During the synaptogenetic period, pharmacological blockade of N-methyl-d-aspartate (NMDA)-type glutamate receptors as well as stimulation of GABAA receptors has been reported to be associated with increased apoptosis in the developing brain. Importantly, recent data suggest that even low, non-apoptogenic concentrations of anaesthetics can perturb neuronal dendritic development and thus could potentially lead to impairment of developing neuronal networks. The extrapolation of these experimental observations to clinical practice is of course very difficult and requires extreme caution as differences in drug concentrations and exposure times as well as interspecies variations are all important confounding variables. While clinicians should clearly not withhold anaesthesia based on current animal studies, these observations should urge more laboratory and clinical research to further elucidate this issue.

Sequential activation of p75 and TrkB is involved in dendritic development of subventricular zone-derived neuronal progenitors in vitro.

Dendritic arbor development of subventricular zone-derived interneurons is a critical step in their integration into functional circuits of the postnatal olfactory bulb. However, the mechanism and molecular control of this process remain unknown. In this study, we have developed a culture model where dendritic development of purified subventricular zone cells proceeds under serum-free conditions in the absence of added growth factors and non-neural cells. We demonstrate that the large majority of these cells in culture express GABA and elaborate dendritic arbors with spine-like protrusions but they do not possess axons. These neurons expressed receptors for neurotrophins including p75, TrkB and TrkC but not TrkA. Application of exogenous neurotrophins, including brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT3) and nerve growth factor (NGF), to cultures stimulated dendritic growth and led to more complex dendritic arbors during the initial 3 days in culture. Our results suggest that these effects are independent of Trk receptors and mediated by the p75/ceramide signaling pathway. We also show that brain-derived neurotrophic factor is the only neurotrophin that is able to influence late-phase dendritic development via TrkB receptor activation. These results suggest that dendritic arbor development of subventricular zone-derived cells may be regulated by neurotrophins through the activation of p75 and the TrkB receptor signaling pathways in a sequentially defined temporal pattern.

The "rete mirabile" of the clivus and the dorsum sellae. A microanatomical study.

The osteofibrous space underneath the dura mater of the dorsoclival area was studied with respect to the arterial branches arising from the internal carotid artery at this level. Special attention was given to the main variations of branching and anastomosing patterns found in this area. Our results indicate that the meningohypophyseal trunk is the main supplier of the dorsoclival area. The dorsal meningeal artery was present in all cases. In the course of this vessel three main variations were observed. The inferior hypophyseal and the tentorial artery also contributed to the arterial supply of this region. A large number of anastomoses between the internal and external carotid artery was present as well. Because of the refinement of microsurgical and interventional techniques pathological lesions like tumors and vascular malformations of the dorsoclival area are frequently accessible. The knowledge of the microvascular anatomy of the clivus and the dorsum sellae is important as the rich collateral network found in this area may contribute to the arterial supply of pathological lesions arising here.

The meningohypophyseal trunk and its blood supply to different intracranial structures. An anatomical study.

50 meningohypophyseal trunks of the intracavernous internal carotid artery were studied in 25 human cadavers. Special attention was given to the main variations of branching patterns of this trunk. The dorsal meningeal arteries were found in all cases and three typical variations were observed in their origins and courses: a prominent medial branch (52%), a bifurcating type (38%) or a single lateral branch was present (10%). The tentorial artery of Bernasconi-Cassinari arised as a single branch in 64% of the cases, while in 36% two or more branches took a direct origin from the main trunk. The inferior hypophyseal artery was prominent in 82% of the cases. The inferolateral trunk had a common origin with the meningohypophyseal trunk in 8% of our dissections. A large number of anastomoses between these vessels was observed. As a number of tumorous and vascular pathologies, which can be treated with microsurgical techniques, take their origin from the cavernous sinus, the knowledge of the smaller vessels arising from the intracavernous internal carotid artery as well as their main variations is important.

Removal of PSA from NCAM affects the survival of magnocellular vasopressin- and oxytocin-producing neurons in organotypic cultures of the paraventricular nucleus.

The expression of the polysialic acid neural cell adhesion molecule (PSA-NCAM) in the hypothalamo-neurohypophyseal system has been correlated with morphofunctional plasticity. In this study, we investigated the role of PSA-NCAM in the survival of oxytocin (OT)- and vasopressin (VP)-producing magnocellular cells of this system. We used a recently developed organotypic slice culture model of the rat hypothalamic paraventricular nucleus (PVN) in which ciliary neurotrophic factor (CNTF) and leukemia inhibitory factor (LIF) are potent survival factors for magnocellular neurons. We demonstrate by means of confocal microscopy that cultured magnocellular VP and OT neurons express strong immunoreactivity for PSA-NCAM. Removal of PSA from NCAM by the enzyme Endo N leads to a significant loss of both VP and OT neurons in the presence of low concentrations of CNTF. Endo N treatment did not change cell survival in the presence of LIF. These results suggest that, in addition to its role in neuro-glial plasticity, PSA-NCAM might also influence the trophic factor responsiveness of hypothalamic VP and OT neurosecretory cells.

Differential neurotoxic effects of propofol on dissociated cortical cells and organotypic hippocampal cultures.

BACKGROUND: Propofol is a widely used anesthetic agent for adults and children. Although extensive clinical use has demonstrated its safety, neurologic dysfunctions have been described after the use of this agent. A recent study on a model of aggregating cell cultures reported that propofol might cause irreversible lesions of gamma-aminobutyric acid-mediated (GABAergic) neurons when administered at a critical phase of brain development. We investigated this issue by comparing the effects of long-term propofol treatment on two models of brain cultures: dissociated neonatal cortical cell cultures and organotypic slice cultures. METHODS: Survival of GABAergic neurons in dissociated cultures of newborn rat cortex (postnatal age, 1 day) treated for 3 days with different concentrations of propofol was assessed using histologic and cytochemical methods. For hippocampal organotypic slice cultures (postnatal age, 1 and 7 days), cell survival was assessed by measuring functional and morphologic parameters: extracellular and intracellular electrophysiology, propidium staining of dying cells, and light and electron microscopy. RESULTS: In dissociated neonatal cell cultures, propofol induced dose-dependent lesions of GABAergic neurons and of glial cells. In contrast, no evidence for neurotoxic effects of propofol were found after long-term treatment of organotypic slice cultures. Excitatory transmission was not affected by propofol, and inhibitory transmission was still functional. Histologic preparations showed no evidence for cell degeneration or death. CONCLUSION: Although long-term applications of propofol to dissociated cortical cell cultures produced degeneration and death of GABAergic neurons and glial cells, no such lesions were found when using a model of postnatal organotypic slice cultures. This conclusion is based on both functional and morphologic tests.

The role of neural cell adhesion molecules in plasticity and repair.

Repair and functional recovery after brain injury critically depends on structural and functional plasticity of preserved neuronal networks. A striking feature of brain structures where tissue reorganization and plasticity occur is a strong expression of the polysialylated neural cell adhesion molecule (PSA-NCAM). An important role of this molecule in various aspects of neuronal and synaptic plasticity has been revealed by many studies. Recently, a new mechanism has been elucidated whereby PSA-NCAM may contribute to signalling mediated by the neurotrophic factor BDNF, thereby sensitizing neurons to this growth factor. This mechanism was shown to be important for activity-induced synaptic plasticity and for the survival and differentiation of cortical neurons. A cross-talk between these molecules may, thus, reveal a key factor for properties of structural plasticity and in particular could mediate the activity-dependent aspects of synaptic network remodeling. Animal models have been developed to assess the role of these molecules in functional recovery after lesions.